Ribosomal protein synthesis is a discontinuous process, with pauses modulating the rate of translation. Such pauses can be crucial for the successful completion of co-translational events such as protein folding and side chain modification4. Codon and codon pair usage, internal Shine-Dalgarno sequences and downstream mRNA secondary structures are a few of the known translational pausing elements. However, the magnitude to which these elements induce pausing has not yet been quantified. In this proposal, the pausing elements stated above will be evaluated for their effect on the rate of translation. Because translation elongation is a stochastic process, it is impossibl to synchronize ribosomes for ensemble studies. To bypass this limitation, single molecule fluorescence resonance energy transfer (smFRET) experiments will be performed and visualized by total internal reflection fluorescence (TIRF) microscopy in order to follow the translation rate of single ribosomes. In these experiments, FRET signals are transiently generated when Phe-tRNAPhe (labeled with a FRET acceptor dye) is delivered to the ribosome (labeled with a FRET donor dye) during the translation of EmGFP mRNA. The FRET signals are then assigned to the respective Phe residues within the EmGFP protein sequence to define discrete translated segments, and the translation time of each segment is quantified. Using this platform, pausing elements will be inserted into the segments of EmGFP to determine their effect on translation rate. These studies will shed light on the magnitude to which these elements affect the rhythm of translation elongation. In addition, these studies should also shed light on the potential effects of silent synonymous mutations (mutations that change the codon identity but not the amino acid it codes for), which have been associated with protein misfolding and changes in physiology.